3-hydroxy-11-ketoetiocholanic acid and its esters



Patented May 29, 1951 UNITED STATES PATENT OFFICE 3-HYDROXY-1I-KETOETIOCHOLANIC ACID AND ITS ESTERS Tadeus Reichstein, Basel, Switzerland No Drawing. Original application February 4,

1943, Serial'No. 474,126. Divided and this application February 27, 1946, Serial No. 650,733. In Switzerland April 25, 1942 5 Claims.

It has been found that compound or" the cyclopentanop-olyhydrophenanthrene series containing oxygen, or groups including oxygen, in ring C, can be produced by treating compounds of this series, containing a nuclear double bond in which the carbon atom 11 participates, with agents capable of eliminating this double bond with production of addition compounds, in which at least one of the valencies having taken part in the double bond is occupied by oxygen and, if de- Patent No. 2,409,798 from the corresponding com- 1 pounds containing, for instance in the 12 position, a free or substituted hydroxyl group; or by degradation of the side chain as described in U. S. Patent No. 2,387,705. The remaining rings of the nucleus are saturated or unsaturated. The starting products may further be ubstituted in anyway, e. g. in the 3, 7 and/or 17 position. The following compounds may for example be used: (3 -3 hydroxy cholenic acid, A 3-ketocholenic acid, A -3,7-dihydroxy-cholenic acid, it;

A -3-hydroxycho1dienic acid, A -3- keto-choldienic acid, A -pregnen-3,20-dione, A -pregnadiene 3,20-dione, A -.etiochlens-3,17-dione, A etio choladiene-3,l7- dione, the lower homologues of the above acids as, for example, A -S-hydroxy-etio-cholenic acid or A -3-keto-etiocholenic acid or the corresponding nor-cholenic acid or bisnor-cholenic acid. The corresponding derivative esterified in the hydroxyl and/0r carboxyl groups and analogous compounds unsaturated in the 9,11 position may also be used.

The starting materials containing a double bond in ring C are treated, according to the present process, with agents capable of forming, with removal of this double bond, addition compounds in which at least one of the newly available valencies is occupied by oxygen. In particular therefore, oxidizing agents are used which are capable of adding oxygen or groups including oxygen to the double bond, e. g. peroxides such as hydrogen peroxide or per-acids, metal oxides such as osmium tetroxide or vanadic acid, if .desired in the presence of chlorates; further permanganates, lead tetra-acylates, aryl iodosoacylates or a halogen-silver benzoate complex. Typohalogenous acids, their salts, ethers or esters, or substances'which give off hypohalogenous acids in the presence of water, e. g. bromacetamide or toluene-sulpho-chloramine, may be used instead of the oxidizing agents mentioned, particularly when startin from 11,12 unsaturated compounds.

Halo-hydrins obtained can be subsequently converted into oxides by the action of substances which split off hydrohalide, e. g. alkaline substances, in particular aluminum oxide. In this way oxides are generally obtained which are stereoisomers of those prepared directly by the action of peroxides on the unsaturated starting materials.

Oxide groups can be split up with reducing agents e. g. with amalgams, with metal alcoholates or phenolates such as those of aluminum or magnesium, in the presence of secondary alcohols, with alkali metals and alcohols, with catalytically activated (e. g. by metals) hydrogen, or by electrolytic or biochemical means. Oxide groups may also be converted directly into keto groups by using the known methods which effect isomerization of an oxide into the keto form, e. g. with dilute aqueous or alcoholic acids or alkalis, zinc chloride or concentrated acids such as phosphoric acid.

Halogen atoms which have been introduced, as for example in the halohydrins, can also be removed by means of suitable reducing agents, e. g. zinc and glacial acetic acid. Advantageously, prior to this reduction adjacent free hydroxyl .groups which may be present, are converted into keto groups. The oxidizing or equivalent dehydrogenating agents suitable for this purpose are well known.

The products may subsequently be further converted in a known way, by esterification, saponification, oxidation or dehydrogenation, reduction, halogenation, elimination of acid, or a combination of these reactions in any order of succession. For example partially esterified glycols, e. g. glycol-mono-acylates or halo-hydrins may be directly converted in known way into ketones, by treatment with agents eliminating acid, such as finely divided-metals (in particular zinc) in inert diluents, or with suitable reagents which eliminate hydrohalide. The formation of ketones in this reaction presumably is attributable to the already mentioned isomerizing ability of ring oxides intermediately formed.

By the present process there may be thus obtained the following intermediate and final products: Compounds of the cyclopentanopolyhydrophenanthrene series (among others saturated ones in which the rings A and B are linked together in cis-position), which contain, instead of the initial double bond in ring C, as sole substituent in this ring and preferably, in ll-position a keto group, or free or esterified hydroxyl group. Compounds of this type have been described formerly in the literature as obtained by transformation and degradation or ag lucones: with cardiac activity like digoxigenin. It has been shOWn recently that the original hydroxyl group in ring C of the latter compounds isnot- Example 1 2g. of A -cholenic acid methyl-ester (obtained in platelets, 111. pt. 56-58 C. or in needles m. pt. 61 C., for example from 12-hydroxy-cholanic acid methyl-ester, m. pt. ll9l2l C., by benzoylation and subsequent elimination of benzoic acid at 260-340 C.) are dissolved in 90 cc. of tertiary butyl alcohol, and 40 cc. of water added to the solution. 1.484 g. (2 mols) of bromacetamide are added and the solution allowed to stand in the dark whereby it becomes yellow after a few hours and is coloured dark brown on the next day. It is decolourized by the addition of 4.35 cc. N/ thio-sulphate solution and the majority of the butyl alcohol distilled ofi in vacuo at a bath temperature of 40 C. The suspension is extracted three times with ether, theethereal solution washed with very dilute thio-sulphate solution, sodium carbonate solution and water, dried and evaporated. The residue obtained is dissolved in 24 cc. of glacial acetic acid, and 24 cc. of a 2% solution of chromic acid (480 mg. ClOs) are added. After standing overnight, the greenish brown solution is diluted with. 500 cc. of water and then extracted three times with a large quantity ofether. The ether solutions are washed until neutral, dried and evaporated down. The residue is dissolved in 20 cc. of glacial acetic acid and heated for 30 minutes on a boiling water bath with 3 g. of zinc dust, stirringfrequently. The solution is then evaporated to dryness in vacuo, the residue repeatedly extracted with ether and the ether solution washed with water, sodium carbonate solution and water, dried and evaporated down. The residual crude product is dissolved in petroleum ether and chromatographed through 90 g. of aluminium oxide.

For elution, petroleum ether and then a mixture of benzene and petroleum ether are used, when unchanged parent material is at first obtained and afterwards fractions yielding prisms of melting point 88-89 C. and the specific rotation [a]D =+46.0:1 (concentration 2.024 in acetone). This substance is ll-keto-cholanic acid methyl-ester. From the last fractions extracted with mixtures of benzene and petroleum ether, A -l2-keto-cholenic acid methyl-ester can be isolated, which, after recrystallization from methyl alcohol, melts at 88-90 C.

Acetone for example may also be used as solvent instead of tertiary butyl alcohol. Addition of for example glacial acetic acid accelerates the 4 reaction, which can alsobe carried out in hetero geneous phases, e. g. inetherandwater.

Example 2 100. mg. of A -FChOIBHiC acid methyl-ester are boiled for minutes with 1.55 cc. of a 2 per cent solution of caustic potash in methyl about 3-molecules of hypobromous acid.)

alcohol, with addition of 0.1 cc. water. The methyl alcohol is then removed in vacuo, the solution acidified and extracted three times with ether. The ether solutions are washed with waterydriedand evaporated down. The residue is dissolved in 7 cc. of tertiary butyl-alcohol, 0.13 cc. of 2 N caustic soda solution added and then N/ 10 caustic soda solution until a faint pink colouration is obtained with phenolphthalein. After the excess of alkali has been removed by running in carbon-dioxida a freshly filtered solution of hypobromous acid, prepared at 0 C. from 0.08 cc. bromine, about 1 cc. of water and an excess of silver carbonate, is added at 0 C. and the mixture allowed to stand in an ice box for 20 hours. (The acid solution contains, as was established by a preliminary experiment There is no separation of bromine. After the addition of some sodium bisulphite solution the mixture is weakly acidified with hydrochloric acid, diluted with plenty of water and extracted three times with ether. The ether solutions are washed with hydrochloric acid and water, dried, concentrated, and an excess of diazomethane solution is added. After 5 minutes the solution is worked up in the usual way, 1.5 cc. of glacial acetic acid and 1.5 cc. of a 2% chromic acid solution are added to the crude product, and the whole. is allowed to stand for 46 hours. The oxidation product is then dibrominated with 500 mg. of zinc dust in 5 cc. of glacial acetic acid by heating for 15 minutes on a water bath. The crude product obtained in this way is dissolved in petroleum ether and chromatographed through a column containing 3 g. aluminium oxide, when the ll-keto-cholanic acid methyl-ester of M. pt. 8889 C., described in Example 1, can be isolated.

Instead of hypobromous acid there may be used its esters, ethers or salts, for example the methyl or ethyl'ester, or the hypobromous acid may be used in presence of acetic acid whereby an acetylated halohydrin is obtained.

Example 3 mg. of A f- -cholenic acid methyl-ester are dissolved in- 4.5 cc. of tertiary butyl alcohol and 0.5 cc. glacial acetic acid. A solution of 151.5 mg. of the sodium salt of toluene-sulfochloramine in 2 cc. of water is added, and the whole allowed to stand for 24 hours. The solution is then diluted with much water, extracted with ether, the ethereal solution washed until neutral, dried and evaporated down. The residue is allowed to stand overnight in 1.5 cc. glacial acetic acid with 1.5 cc. of a 2% chromic acid solution. The oxidation product, after working up, is dechlorinated by heating for 45 minutes with 500 mg. of zinc dust in 5 cc. of glacial acetic acid. The crude product obtained in this way gives, on chromatography, in an analogous way to that described in Example l, the ll-keto-cholanic acid methyl-ester, M. pt. 88-89 C.

100 mg. of l1- -keto-cholanic acid methyl-ester are hydrogenated in 4 cc. of glacial acetic acid with 55.4 mg. platinum oxide. After hydrogenation is complete, the platinum is filtered ofi, the filtrate evaporated down in vacuo, the

residue dissolved in ether and the ether solution washed until neutral, dried and evaporated down. The crude product obtained is first recrystallized from petroleum ether, then from methyl alcohol with the addition of a little water. In this way bunches of needles are obtained which after washing with diluted methyl alcohol and drying melt at 87-88 C. The mixed melting point with the keto-ester is 60-77 C. and the specific rotation [a]D =+49.8i2 (concentration 1.505 in acetone). The product is an ll-hydroxy-cholanic acid methyl-ester.

Instead of platinum oxide and glacial acetic acid the reduction can also be carried out with Raney nickel in methyl alcohol.

Example 4 200 mg. of A -3-keto-cholenic acid methylester, M. pt. 122-124 C. (obtained by the process described in U. S. Patent application Serial No. 433,072) are dissolved in 12 cc. of tertiary butyl alcohol (or 16 cc. acetone). To this solution a solution of 140 mg. of N-bromacetamide in 4 cc. of water is added and the whole allowed to stand for 16 hours at room temperature, when 1.6 mols of the agent are used. After diluting with water the mixture is concentrated down in vacuo, the suspension extracted with ether, the ether solution washed with sodium carbonate solution and water, dried over sodium sulphate and evaporated down. The crude residue is then dissolved in 4 cc. of glacial acetic acid, a solution of 48 mg. of chromium trioxide and 2.4 cc. glacial acetic acid added and the whole allowed to stand for 16 hours at 20 C. It is then evaporated down in vacuo at a bath temperature of 25-30 0., water added to the residue, and extracted with ether. The ether solution is washed with dilute sulphuric acid, sodium carbonate solution and water, dried over sodium sulphate and evaporated down. The residue is dissolved in 4 cc. of glacial acetic acid, 150 mg. of zinc dust added and the whole heated for minutes on a boiling Water bath, shaking continually. The solution is again evaporated down in vacuo, water is added to the residue, and the mixture extracted with ether. After washing the ether solution with dilute hydrochloric acid, sodium carbonate solution and water, and drying over sodium sulphate, it leaves a residue on evaporating down which is dissolved in 1 cc. of absolute benzene and 19 cc. of petroleum ether. This solution is chromatographed by allowing to run through about 6 g. of aluminium oxide. The fractions obtained by elution with mixtures of benzene and petroleum ether and the first ones obtained with pure benzene give the unchanged parent material, M. pt. 122 C. Further fractions extracted with benzene give, after recrystallization from ether-petroleum ether mixtures, 3,11-diketo-cholanic acid methyl-ester of the formula OOCH:

specific rotation [a]D =72i2 (concentration 1.22 in acetone). The ultraviolet absorption spectrum in alcohol shows a maximum at 238 m,u(log e=3.92)

Example 5 1 g. of A -3-acetoxy-cholenic acid methylester of M. pt. 117-118 0., (obtained for example as described in U. S. patent application Serial No. 433,072) is dissolved in 100 cc. of acetone, a solution of 720 mg. N-bromacetamide in 20 cc. of water added and the Whole allowed to stand at 20 C. for 16 hours. More water is then added, the acetone removed in vacuo, the residue extracted with ether, the ether solution washed with a solution of sodium carbonate and water, dried over sodium sulphate and evaporated down. The crystalline residue is dissolved in 5 cc. of chloroform and 5 cc. of glacial acetic acid, 20 cc. of a 2% solution of chromium trioxide in glacial acetic acid added and the whole allowed to stand at 20 C. for 16 hours. After removal of the glacial acetic acid in vacuo at a bath temperature of 30 C., water is added to the residue, the whole is extracted with ether, the ether solution washed with dilute sulphuric acid, sodium carbonate solution and water, dried over sodium sulphate and concentrated down. The residue is dissolved in 8 cc. of glacial acetic acid, 0.7 g. of zinc dust and 0.5 g. of sodium acetate added and the whole heated for 15 minutes on a boiling water bath, shaking continually. The mixture is then filtered and the filtrate evaporated down in vacuo. Water is added to the residue, the mixture extracted with ether, the ether solution washed with dilute hydrochloric acid, sodium carbonate solution and water, dried over sodium sulphate and evaporated down. The crystalline resin obtained in this way melts in the crude state at -130 C. It is separated by chromatographing over 30 g. of aluminium oxide. The fractions obtained :by elution with petroleum ether and with a mixture of benzene and petroleum ether (1:2) yield the unchanged parent material, M. pt. l17-118 C.

The fractions extracted with benzene-petroleum ether mixture (1:1) and the first fractions extracted with absolute benzene give 3- acetoxy-ll-keto-cholanic acid methyl-ester of' the formula OOCHa 7 specific rotation la] -=+67.1i2 (concentration 1.557 in acetone). The product may be saponified in known manner to the free 3-hydroxyll-keto-cnolanic acid.

The further fractions eluted with absolute benzene and a mixture of benzene and ether give, after recrystallization from acetone, A -3-acetoxy-lZ-keto-chdlenic acid methyl-ester as 001- ourless platelets, melting at 149-150" C. and having a specific rotation [a]D =+102.5i2 (concentration 1.979 in acetone). In the ultraviolet absorption spectrum in alcoholic solution a maximum was found at 241ma (log e=3.91).

In an analogous manner the 3-acetoxy-11- keto-cholanic acid methyl-ester of M. pt. 1'76- 1'78 C. and specific rotation [a]D "=+56.4:2 (concentration 1.0633 in acetone) is obtained in addition to A 'l -3-acetoxy-12-keto-cholenic acid methyl-ester, M. pt. 195 196, from the stereoisomeric A -3-acetoxy-cholenic acid methylester, M. pt. 146-149 C. (of. U. S. patent application Serial No. 433,072).

102 mg. of 3-acetoxy-1l-keto-cholanic acid methyl-ester, of M. pt. 132-133 0., are dissolved in glacial acetic acid and hydrogenated with 74 mg. of platinum oxide. The absorption of gas is practically ended after 6 hours. To be on the safe side, shaking is continued for 24 hours. After filtration, the filtrate is evaporated down in vacuo, the residue dissolved in ether, the ether solution washed with sodium carbonate solution and water, dried over sodium sulphate and concentrated down to a small vol ume. On standing, 3-acetoxy-ll-hydroxy-cholanic acid methyl-ester of the formula crystallizes out in long needles, melting at v (concentration 1.018 in acetone). This product may be esterified also in 11 position by energetic action of esterifying agents.

70 mg. 3 acetoxy 11 keto cholanic acidmethyl-ester, M. pt. 132-133 C., are boiled under reflux for minutes with a solution of 70 mg. of caustic alkali in 4 cc. methyl alcohol. The solution is then diluted with water, the methyl alcohol removed in vacuo, hydrochloric acid is added until an acid reaction to congo is reached, and the precipitated acid is extracted with ether. The ether solution is washed with water, dried over sodium sulphate and evaporated down. The 3-hydroxy-ll-keto-cholanic acid obtained in this way melts in the crude state at 218-223 C. mg. of the methyl-ester prepared from it with the aid of diazomethane are oxidized with a solution of 14 mg. of chromium trioxide in 1.5 cc. of glacial acetic acid for 16 hours. After working up in the usual way, 3,11-diketo-cholanic acid methyl-ester is OOCH:

The same product can also be obtained if, instead of M 3 acetoxy-cholenic acid methylesters of M. pt. 117-118 C. or 146-149 C., the corresponding non-acetylated compounds of M. pt. -l01 C. or 109110 C. are used. In this case the 3,11-diketo compound is obtained didirectly as a result of the oxidizing action of bromacetamide, in place of which other hypohalide derivatives can also be used.

0.5 g. of 3,11-diketo cholanic acid methyl-ester is hydrogenated in 8 cc. of pure glacial acetic acid with the addition of 24 mg. of platinum oxide. During 25 minutes, 39.2 cc. of hydrogen are absorbed (theoretically for 1 mol, 32.3 cc.) and afterwards the absorption continues only very slowly. It is interrupted, the mixture filtered, washed with ether and evaporated down. The residue is dissolved in chloroform and ether, the solution washed with sodium carbonate solution and. water, dried over sodium sulphate and evaporated down in vacuo. By separation of the, reaction mixture thus obtained with digitonin, a 3-hydroxy 11 keto cholanic acid methyl-ester, M. pt. 152-153 C., is obtained as main product, together with the corresponding compound isomeric in 3-position. The ester of M. pt. 152-153 C. gives a 3-acetate of M. pt. 176-178" C. which is identical with that formerly described.

495 mg. of this 3-acetoxy-1l-keto-cholanic acid methyl-ester, M. pt. 176-178 C., are hydrogenated in 8 cc. of pure glacial acetic acid with 98 mg. of platinum oxide. The absorption of hydrogen is almost complete after 8 hours, after which the reaction is activated by shaking up with air, the mixture further hydrogenated and this treatment repeated. Altogether, shaking is continued for 24 hours in an atmosphere of hydrogen. The mixture is then filtered, washed with ether and the filtrate evaporated down in vacuo. The residue is dissolved in much ether, the solution washed with dilute hydrochloric acid, sodium carbonate solution and water, dried over sodium sulphate, and concentrated down to a small volume. The crystals separating out are recrystallized from a large quantity of ether. In this way a 3-acetoxy-11- hydroxy-cholanic acid methyl-ester, M. pt.

139-140 C. and specific rotation (concentration 0.992 in acetone) is obtained, being a stereoisomer of the compound described above.

Example 6 (a) 200 mg. of A -3-keto-etio-cholenic acid methyl-ester, M. pt. 137-139" C. (obtained e. g. by the process described in U. S. pat. appln. Serial No. 433,072 or Patent No. 2,387,706) are dissolved in 16 cc. of acetone, a solution of mg. of N-bromacetamide in 4 cc. of water is added and the whole allowed to stand at room temperature for 16 hours. After the addition of some 'wa.-

(b) To 55 mg. of the crystals melting at '190-192 C. are added 2 cc. of glacial acetic acid and then a solution of 13 mg. of chromium trioxide in 0.65 cc. glacial acetic acid and thewhole allowed to stand for 16 hours at room temperature. It is then evaporated down in vacuo at 30 C., water added to the residue, extracted with ether, the ether solution washed with dilute sulphuric acid, sodium carbonate solution and water, dried over sodium sulphate and evaporated down. The residue gives, from an ether-petroleum ether mixture, crystals of the keto-bromide, M. pt. 171-178 (3., having the formula:

I OH;

COOCH:

(c) 15 mg. of the keto-bromide, M. pt. 171-178 0., are dissolved in a little glacial acetic acid and heated for 15 minutes on a boiling water bath with mg. of zinc dust, shaking continually. The

solution is evaporated down in vacuo, water added to the residue, and the latter extracted several times with ether. The ether solution is washed with dilute sulphuric acid, sodium carbonate solution and water, dried over sodium sulphate and evaporated down. The residue gives, from ether, the 3,11-diketo-etio-ch0lam'c acid methyl-ester, M. pt. 186190 0., having the formula:

compound is [a] =-}-91.1i2 (concentration 1.031 in acetone). The alcoholic solution shows in the ultraviolet absorption spectrum strong selective absorption with a maximum at 239.5 m (log =4.

(d) 34 mg. of 3,1l-dilgeto-etio-cholanic acid mehyl-ester, M. pt. 186-190 C., are dissolved in 0.2 cc. glacial acetic acid. To this'solution 0.2 cc. of a bromine solution in glacial acetic acid is gradually added, which has been prepared by mixing 0.1 cc. bromine with 3.84 cc. glacial acetic acid. After decolourization is complete, the solution is concentrated in vacuo, and the crystals thus separated out are washed with some ether. The product obtained has the formula:

' the residue dissolved in ether, the ethereal solution washed with dilute hydrochloric acid, sodium carbonate solution and water, dried over sodium sulphate and evaporated down. The residue is chromatographed, when the fractions eluted with absolute benzene and with a mixture of benzene and ether yield the A -3,11,diketo-etio-cholenic acid methyl-ester of the'following formula:

C 00 CHa in 16 cc. of pure glacial acetic acid with 30 mg.

platinum oxide. The absorption .of gas is very rapid at the beginning and ceases almost completely after 30 minutes, when the hydrogenation is discontinued It is then filtered, washed with ether, the filtrate evaporated down in vacuo, theresidue dissolved in ether, the solution Washed with dilute hydrochloric acid, sodium carbonate solution and water, dried over sodium sulphate and evaporated down toa small volume. On boiling, 3-hydroxy-ll-keto-etio-cholanic acid methyl-ester crystallizes out in elongated platelets of M. pt. -174 C. By working up the mother liquors a further quantity of this ester is obtained together with the corresponding compound, stereoisomeric in the 3-position, M. pt. 157-159 C.

By acetylation the corresponding stereoisomeric 3-acetoxy-1l-keto-etio-cholanic acid methyl-esters, M. pt. 129-131 0., and 148-151 C. (see Example 7) are obtained. The compound melting at 129131 C. can be saponified to the free 3- hydroxy 11 keto etio cholam'c acid, M. pt. 204-207 C., which can be converted, by acetyla- COOH AcO

By more energetic hydrogenation, the keto group in ll-position may also be transformed into a carbinol group. The'latter can be esterified in known manner.

Example 7 (a) 215 mg. of A -'3-acetoxy-etio-cholenic acid methyl-ester, M. pt. 99-100" ,0. (obtained as described in U. S. Patent No. 2,387,706) are dissolved in cc. acetone. To this solution one of 150 mg. of bromacetamid in 4 cc. of water is added and the whole allowed to stand for 1.6 hours at room temperature. It is then diluted ,with water, the acetone evaporated .off in vacuoand the residue extracted with ether. The ethersolution is washed with sodium carbonate solution and .water, dried oversodium sulphate and evaporated, down. "The residual crude product is .recrystallized from ether. It consists of al.1210- hydrin, :M. pt. 218-222" .C of theiormula:

(b) 110 mg. of this crystallized oxybromide are dissolved in a little chloroform, a solution of mg. chromium trioxide in. 2 ccglacial acetic acid is added, and the whole allowedto stand at room temperature for 16 hours. On working up in the usual way, the .crystalline .keto bromide of the formula:

coo CH3 COOOHa and melting at 148-15? C. is obtained. Specific rotation [al =+98.1:2 (concentration 1.386 In acetone). By saponification with a solution of 1% hydrogen chloride in methyl alcohol, th corresponding free 3-hydroxy-11ket0-et Q-6 Q- 12 lanicgacid methyl-ester; iszobtained whichican'zbe oxidized. with chromium .trioxidednto'. 3,11 ediketoetioecholanic ,acid. methyleester, 1M. .pt. .186-190" (1.1(describedin Example 6 .under. 0) .ior..reduced into a 3,11-dihydroxyeetiocholanic acid' methylester.

From the mother liquors' in (a)-A -'3-acet Xylz-keto-etio-cholenic acid methyl-ester, M. pt. 157-160 C., can be obtained, by oxidation and debromination.

Example 8 To 668 mg. of A -cholenic acid methyl-ester, M. pt. 59-61 C. (prepared :for example from l2-hydroxy-cholanic acid methyl-ester, M. pt. -121 C., by saponification, decomposition by heating in vacuo at 240-300 C. and subsequent methylation) is added asolution of 7.45 mg.,.perbenzoio'acidin40 cc. of chloroform andthe whole allowedtto stand'for 12 hours at roomtemperature. The chloroform is evaporated ofi in vacuo, the'residue dissolved in ether, the solution washed with-sodium carbonate solution and water, dried over sodium sulphate and considerably concentrated; When pentane is added, crystallization takesplace. After filtering off by suction, washing with pentane and drying,11,12-oxydo-cholanic-acid'methyl-ester, .M. pt. 96-.-97 C. is obtained. On-recrystallization frommethyl alcohol it melts in a'pure condition at 97-98" C. 'Saponification with a solution of potassium'carbonate in methyl alcohol gives 11,12-oxydo-cholanic acid, M. pt. -157 C.

In an analogous way A -3-acetoxy-cholenic acid methyl-ester, M. pt. l17'1'l8 0., gives 11,12- oxydo-3-acetoxy-cholanic acid methyl-ester. If one starts from an .esterified :A -3-hydroxycholenic acid, such as A -B-acetoXy-cholenic acid methyl-ester (obtained for example by reduction of .A -3-acetoxyel2 keto-cholenic acid methyl-ester with hydrazine and sodium ethylate) the corresponding 9,11-oxydo "compounds can-.begobtained.

The oxydo compounds'mentionedcmay bejisomerized in'known manner'toketonesand the :latter if-desiredbe reduced to alcohols. .On theqother side the oxide grouping may besplit ,up'by reduction:in a;known mannerto alcohols .and'the lat ter subsequently be oxidized, if desired, '.to :ketones.

Example 9 240.7 mg. of .A -fi-keto-cholenic acid .(obtainable for example from A -3-keto-cholenic acid methyl-ester, M. pt. 122-124 0., by saponification with 2% caustic alkali in methyl alcohol) are dissolved in 6 cc. of pyridine, stable to permanganate. Then 6.50 cc. of N/lO-caustic soda solution are added, so that the solution is just feebly alkaline to phenolphthalein. A solution of 102.2 mg. potassium permanganate (1 mol) in 15 cc. water is then added in eight portions within a period of 2 hours .and the reaction mixture finally allowed to stand for 2 hours. The precipitate is filtered off and washed with amixture of pyridine and water, the clear filtrate acidified with hydrochloric acid while cooling in ice, and the separated precipitate filtered off by suction and thoroughly washed with water. This precipitate is then dissolved in ether, the ether solution washed once again with water, dried, concentrated down considerably, and an excess of diazomethane added. After 5 minutes the solution is'worked up in the usual way and a crude prodnot is obtained which is dissolved in petroleum ether and chromatographed over 6.3 'g. aluminihours under reflux.

parent material, fractions are obtained which, on recrystallization from ether-petroleum ether mixtures and then from aqueous methyl alcohol, give platelets which melt at 105-107.5 C. after marked sintering. [a],, =+63.4i2 (concentration 1.372 in methyl alcohol). This substance is a 3-keto-11,12-dihydroxy-cholanic acid meth lester.

By splitting off water from this compound, for example by distillation with zinc dust, a diketocholanic acid methyl-ester is obtained. Otherwise by mild oxydation or better by dehydrogenation, for example with a ketone in presence of a metal alcoholate or phenolate, the 11,12-dihydroxy compound yields the 3,11,12-triketocholanic acid methyl-ester.

Example 1.3 g. of A -cholenic acid methyl-ester, M. pt. 59-61 C., are dissolved in 50 cc. of absolute ether, a solution of 1 g. of osmium tetroxide in 50 cc. of absolute ether is added and the whole allowed to stand for three days at room temperature. The dark solution is then concentrated down at first on a water bath and then in vacuo. The residue is dissolved in 10 cc. of benzene and 50 cc. of alcohol, a hot solution of 2 g. of potassium hydroxide and 2 g. of crystallized sodium sulphite in 12 cc. of water added hot to the so lution obtained, and the mixture boiled for 3 2 cc. of saturated aqueous saline with 8 cc. of alcohol is then added to the filtered'mixture to produce better separation of the fiocculent precipitate and the whole boiled for another hour whereby the benzene is allowed to distil ofi, replacing it with alcohol saturated with sodium chloride. It is then filtered hot and thoroughly washed out with hot alcohol which is saturated with sodium chloride.

To the alkaline filtrates, which should .have at the most a yellowish colour, water is added, and then, to prevent frothing, acetic acid until the mixture has a faintly acid reaction to litmus. Then they are completely freed from alcohol in a vacuum. Hydrochloric acid is added until the residue has an acid reaction to congo when it is extracted with ether. The ether solution is washed with some water, dried over sodium sulphate, and a solution of diazomethane in ether added until a permanent yellow colouration is obtained. After 10 minutes, it is washed with aqueous hydrochloric acid, sodium carbonate solution and water, dried over sodium sulphate and evaporated down. There remains a light brown residue, which crystallizes after a few hours.

The crystals are purified by dissolving in a mixture of benzene and petroleum-ether (1:4) and filtering the solution through a column of 30 g. aluminium oxide prepared with petroleum ether. The filtrates obtained with benzene-petroleum ether mixtures (1:1) and, in particular, those obtained with absolute benzene give, on evaporating down, residues which crystallize from a little absolute ether by addition of petroleum ether in colourless needles, M. pt. 83-85 C., or in bunches of coarse, pointed needles, melting at 103-104 C. They consist of 11,12-dihydroxycholanic acid methyl-ester, which forms crystal dimers. The specific rotation of the preparation melting at 83-85 C. is [a] =|11.3il (concentration 1.86 in methyl alcohol). The preparation melting at 103-104" C. has an identical specific rotation of [a] =l-1Z.2:t1 (concentra tion 1.973 in methyl alcohol).

Both preparations prove not to be identical with a presumably. stereoisomeric 11,12-dihydroxycholanic acid methyl-ester, which is obtained from A -cholenic acid by treatment with potassium permanganate and subsequent methylation in an analogous manner to that described in Example 9.

mg. of 11,12-dihydroxy-cholanic acid methyl-ester, M. pt. 83.5 C. or 103-104 C. are boiled for 15 minutes with a solution of 8 mg. potassium hydroxide in 0.5 cc. methyl alcohol. Some water is then added, the methyl alcohol removed in vacuo, and the remaining mixture extracted with ether after the addition of hydrochloric acid. The ether solution is washed with water, dried over sodium sulphate, considerably concentrated,

and pentane added. The free 11,12-dihydroxycholanic acid thus obtained crystallizes in small needles, which collect in aggregates, having a M. pt. 211-214 C.

100 mg. of l1,12-dihydroxy-cholanic acid methyl-ester, M. pt. 83 85 C., or 103-104 C. are boiled with 0.6 cc. of acetic anhydride and 1 cc. of pyridine for 8 hours under reflux. The mixture is then evaporated down in vacuo, the residue dissolved in ether, the ether solution washed with hydrochloric acid, sodium carbonate solution and water, dried With sodium sulphate and evaporated down. The residue gives, when recrystallized from methyl alcohol, 11,12-diacetoxycholanic acid methyl-ester in colourless rods, M. pt. 108-1l0 C., [a] =+1.5i1 (concentration 1.94 in acetone). By heating this compound with zine dust in toluene in known manner and subsequent saponification of the product with an alcoholic solution of potassium hydroxide, a ketccholanic acid is obtained, which after treatment with diazomethane yields a keto-cholanic acid methyl-ester.

In instead of A -cholenic acid methyl-ester there are used as starting materials corresponding derivatives, containing an additional hydroxyl, acyloxy or keto group in 3-position of the cholene or etiocholene nucleus, correspondingly substituted products of the said series are obtained.

Example 11 300 mg. of A -cho1enic acid methyl-ester are dissolved in 60 cc. of acetone, a solution of 222 mg. bromacetamide (2 mols) is added and the whole allowed to stand for 16 hours at room temperature. The acetone is removed in vacuo, Water added to the residue and the whole extracted with ether. The ether solution is washed with sodium carbonate solution and water, dried over sodium sulphate and evaporated down. The residue is treated in a column as used for chromatography by aluminium oxide. At first a dibromide, M. pt. l01.5-103.5 C. is eluted with petroleum ether. The fractions extracted afterwards with petroleum ether and a mixture of petroleum ether and benzene (3:7) give, on recrystallization from aqueous methyl alcohol, the a-oxide, M. pt. 64.5-65.5 0., which has the formula:

Further eluates probably contain the 9,11-dibromo-l2-hydroxy-cholanic acid methyl-ester, as debromination of this product with zinc dust and subsequent oxidation with chromic acid yield the A -12-keto-cholenic acid methyl-ester.

The same a-oxide can also be obtained by treating the crude product of the bromacetamide reaction with zinc dust or other agents which eliminate hydrohalide.

86 mg. of the a-oxide described are dissolved in 3 cc. of methyl alcohol, the quantity of Raney catalyst obtained from 300 mg. of alloy is added, and hydrogenation performed during 2 hours at 100 C. and 120 atmospheres in a rotating autoclave. After filtration the solution is evaporated down, the residue dissolved in ether and the ether solution washed with sodium carbonate solution and water, dried over sodium sulphate and evaporated down. The residue is chromatographed over aluminium oxide. The fractions extracted with petroleum ether crystallize from methyl alcohol and thus give a small quantity of cholanic acid methyl-ester. The ll-hydroxycholanic acid methyl-ester, M. pt. 87-88 C., already described in Example 3, is eluted with mixtures of benzene and petroleum-ether in the concentrations 1:9 till 3:7. It has the formula:

COOCH;

Example 12 500 mg. of A -3-keto-cholenic acid methylester, M. pt. 120-122 C., are dissolved in 40 cc. of acetone, a solution of 350 mg. of bromacetamide (2 mols) in 10 cc. of water is added and the whole allowed to stand for 16 hours at room temperature. After the addition of water, the acetone is removed in vacuo and the residue extracted with ether. The ether solution is washed with dilute sodium carbonate solution and water, dried over sodium sulphate and evaporated down. The residue is treated according to the chromatographic method with 20 g. of aluminium oxide. The eluates with, benzene and benzene-ether mixtures (50:1) give the 3-keto-l1,l2-dibromcholanic acid methyl-ester in colourless needles, which melt after recrystallization from etherpetroleum ether, at 136 138 C. By debromination they yield the parent substance. Further fractions extracted with benzene-ether mixtures of increasing ether content, and with pure ether, give the 11,12-a-oxido-3-keto-cholanic acid methyl-ester which melts, on recrystallization from i6 petroleum ether, at 122-424: C. It has the formula:

, cm I a/\l l I The further fractions eluted with ether-methyl alcohol (9: 1) give on oxidation with chromic acid and debromination with zinc dust, A -3,12-diketo-cholenic acid methyl-ester.

The oxide described yields, on hydrogenation, a reaction product which forms, after acetylation with acetic anhydride and pyridine at room temperature, in addition to the 3-acetoxy-11-hydroxy-cholanic acid methyl-ester, M. pt. 146- 148" C., described in Example 5, the ester which is stereoisomeric in 3 position and shows a M. pt. of 141-442 C., and a specific rotation [a] +50 (acetone).

Example 13 (a) 650 mg. of A1142 pregnene 3,20 dione, M. pt. 13l-l33 C. (obtainable for example from pregnene-l2-ol-3,20-dione, M. pt. 182-184" C., by benzoylation and subsequent elimination of benzoic acid) are dissolved in cc. of acetone; 0.6 g. of N-bromacetamide and 40 cc. of water is added, and the whole allowed to stand for 15 hours at 20 C. The initially colourless solution becomes yellow after a few hours, but is again colourless after 15 hours. After the addition of some water the acetone is removed in vacuo, when the majority of the organic material is deposited on the sides of the flask as a resinous mass. The water is poured off and the resin washed twice with a little fresh water. The washings are twice extracted with a large volume of ether and a little absolute ether added to the resin alone, when considerable quantities 0f crystals remain undissolved; these are filtered off by suction, well washed with ether and dried in vacuo. They are mostly triangular, colourless platelets which melt with decomposition at about 238-245 C. They consist of the halohydrin of the formula:

acetic acid, and then 5 cc. of a 2% solution of are furthermore added to the residue and the whole is again allowed to stand at 20 C. If no 'more free chromic acid is detectable after 3 hours, 2.5 cc. of the 2% solution are again added and this procedure is repeated until chromic acid can be definitely detected afterstanding for 3 hours. Altogether about 21 cc. of the 2% solution (420 mg. 0103) are necessary. Onworking up in the usual way 12-brom-pregnane-3,11,20- trione is obtained as colourless platelets,.M. pt. 176-184 C.

(c) 240 mg. of this keto bromide are dissolved in cc. of glacial acetic acid, 400 mg. of zinc dust added and the whole heated for 15 minutes to .80? C., rotating continually. By filtration, evaporation of the filtrate in vacuo, addition of water 'to the residue and extraction with ether, pregnane-3,ll,20-trione is obtained as bunches of needles, M. pt. 154-156 C.

The mother liquors of stage (a) of the reaction are oxidized as described in (b) and the oxidation product is debrominated together with the mother liquors of stage (b) of the reaction. In this way some A -pregnene 3,l1,20-trione, pt. 184- 186 C., can be obtained in addition to a further quantity of pregnane 3,11,20-trione.

The latter compound may be reduced, e. g. by

catalytic hydrogenation, to the pregnane3,11,20-

triols and these, if desired, esterified and hereupon partially saponified in 3 and position. By oxidation of the ll-monoester, for example with chromic acid, the pregnane-3,20-di'one-11- ole-ester is obtained. This compound in free form, may be obtained directly, e. g. by'partial oxydation or better dehydrogenation of the triols.

(d) 66 mg. of pregnane-3,11,20-trione are dissolved in 2 cc. of pure glacial acetic acid, a drop of hydrogen bromide in glacial acetic acid is added and then gradually a solution of 31.4 mg. bromine in 1 cc. of glacial acetic acid while rotating; the bromineis used up almost immediately. After decolourization the solution is evaporated to dryness in vacuo at a bath temperature of 20 C., a little ether added to the residue and the latter again well dried in vacuo. After again dissolving in absolute ether, bunches of needles soon separate out, which melt, after washing with ether, at 158-160 C. They consist of 4-brom-pregnane-3,l1,20-trione.

(e) 127 mg. of this bromide are boiledfor 6 hours under reflux with 2 cc. of absolute pyridine. After evaporating down in vacuo, the residue is dissolved in ether, the solution washed with dilute hydrochloric acid, sodium carbonate solution and water, dried over sodium sulphate and evaporated down. It is then distilled in a high vacuum at a bath temperature of 170 C. The distillate crystallizes from ether. The granules obtained are further purified by chromatography over a small column of l g. aluminium oxide. The first fractions extracted with a mixture of benzene and petroleum-ether (1:4) give still impure crystals. The further eluates obtained with benzene-petroleum ether, and with benzene yield, on recrystallization from ether, colourless rods melting at 173-1'75 C.

[alD =+243.5:6; [a]54s1 =+283:t6 (concentration 0.382 in acetone). The ll-ketoprogesterone prepared from corticosterone (Helv.

i8 Chim. Acta, vol. 23, page 684 [1940]) of the formula shows under the same conditions an identical melting point and the same specific rotation. The mixed melting point shows no depression.

EmampZe 14 The preparation of the parent material used in this example can be carried out as described in the process of U. S. Patent application Serial No. 433,072 or in the following way:

A -3-hydroxy-etio-cholenic acid methyl ester is saponified, acetylated, converted into the acid chloride with thionyl chloride, and this acid chloride allowed to react with an excess of an ether solution of diazomethane. The crude acetylated diazoketone is saponified with methyl alcoholic caustic soda at room temperature and the hydroxy diazoketone obtained converted, by heating with glacial acetic acid, in A -21-acetoxypregnene-3-ol-20-one. The latter is converted into A -21acetoxy-pregnene-3,20-dione by boiling with benzene, acetone and aluminium phenolate or by allowing to stand with approximately the calculated quantity of chromic acid in glacial acetic acid. This compound can be purified by chromatography or by, recrystallization from ether. For the following reaction, however, crude products which do not have a sharp melting point, can also be used.

1 part of this A -21-acetoXy-pregnene-3,20- dione is dissolved in 25 parts of acetone, a solution of 0.75 parts of N-bromacetamide in 6' parts of water is added and the whole allowed to stand for 16 hours at room temperature. About 6 parts of water are then added, the acetone removed in vacuov and the residue extracted several times withaether. ..The ether solution, after washing with water and sodium carbonate solution, is

dried and evaporated down. The crystalline residue is dissolved in 18 parts of very pure glacial acetic acid, 18 parts of a solution containing 2% of chromium trioxide added and the whole allowed to stand for 16 hours at 18 C. Two parts of 2% chromium trioxide solution are then again added and the procedure repeated until some chromic acid is detectable in a sample of the solution after a reaction time of 5 hours. The solution is then evaporated down to a small volume at a bath temperature of 30 0., water is added and extraction with ether performed. 0.6 part of zinc dust and 0.5 part of anhydrous sodium acetate are added to the ether solution, the latter concentrated down, and 3 parts of glacial acetic acid added to the residue. The whole is heated for 15 minutes on a boiling water bath, rotating continually. It is then filtered, washed with ether, the filtrate evaporated down in vacuo, water added to the residue and the Whole extracted with a large volume of ether. The other solution, after washing with dilute hydrochloric acid, sodium carbonate solution and water and drying over sodium sulphate gives 0.9-1 part of a crude product, which is purified by chromatography. In particular the fractions eluted with absolute benzene crystallize from ether-petroleum 19 ether mixtures in colourless needles, melting at l52-154 C. The product has a specific rotation [a]D =+107 (acetone), and reduces alkaline silver diamine solution rapidly and effectively at room temperature. It consists of 21-acetoxypregnane-3,11,20-trione.

The same compound can be obtained from A -21-acetoxy-pregnene-3-ol20-one (which is itself obtainable from A -3-acetoxy-etio-cholenic acid via the acid chloride and diazoketone) by reacting directly with 3 mols of bromacetamide, oxidizing and debrominating. The simplest procedure is to start directly from the mixture of compounds isomeric in 3-position, as it is obtained from the product arising by reduction of the 3-keto-acid ester. If, however, one starts from a derivative containing an esterified hyroxyl in 3-position, the corresponding products, containing in this position an esterified or free hydroxyl may be obtained.

By mixing 10 parts of bromide with 384 parts of glacial acetic acid an N/l-bromine solution is prepared; to 9.6 parts of the product, M. pt. 152-l54 C., dissolved in 50 parts of glacial acetic acid, 2 drops of the above bromine solution are added. After a few minutes the solution is suddenly decolourized after which 48 parts of the bromine solution are slowly added, cooling and rotating, and are decolourized almost instantaneously. After evaporating down the solution in vacuo at a bath temperature of 25 C. crystallization occurs. The colourless crystals melt, after washing with ether, at ISO-185 C. The consist of 4-brom-pregnane-3,11,20-trione- 21-ol-acetate.

This bromide is boiled under reflux for 5 hours with 100 parts of absolute pyridine. After evaporating down in vacuo, the residue is dissolved in a large volume of ether, the ethereal solution washed with a little dilute hydrochloric acid, sodium carbonate solution and water, dried over sodium sulphate and evaporated down. The residue is purified chromatographically over a column of aluminium oxide, when the first fractions extracted with benzene give crystals with a low indefinite melting point. The further eluates obtained with benzene, and benzeneether mixtures yield, after recrystallizing twice from acetone-ether, colourless needles, melting at 175-178 C. and showing a specific rotation [a] =+2lli3 (concentration 0.676 in acetone). They consist of A -pregnene-3,11,20- trione 21 ol acetate (dehydro corticosterone acetate). A sample of very pure natural dehydro-corticosterone acetate melts under the same conditions at 177179 C., shows no depression of the melting point in mixture with the synthetic product, and has the same specific rotation.

By saponifioation with a methyl alcoholic solution of hydrogen chloride or an aqueous methyl alcoholic solution of potassium bicarbonate, free dehydro corticosterone, M. pt. 174-180 C. is obtained. The latter may subsequently be converted in known manner into any ester such as the propionate, palmitate, benzoate, succinate, the butyrates, phosphates or carbonic acid esters.

The 21-acetoxy-pregnane3,11,20-trione described or a corresponding ester may be hydrogenated to the 3,11,20,21-triol-21-monoesters and then partially reoxidized or redehydrogenated, as described in Example 18, to the pregnane-ll,21- diol-3,20-dione-21-monoesters. The latters finally may be esterified in 11 position also.

This application is a division of my application Serial No. 474,726, filed February 4, 1943, now Patent No. 2,403,683.

What I claim is:

1. A compound selected from the group consisting of a saturated etiocholanic acid and the corresponding saturated etiocholanic acid methyl ester, the rings A and B of which compound are linked together in cis-position, and which compound contains a hydroxyl at the carbon atom 3 and a ketonic oxygen as sole oxygenic substituent in ring C at the carbon atom 11.

2. A saturated etiocholanic acid methyl ester, the rings A and B of which are linked together in cis-position, and which contains a hydroxyl at the carbon atom 3 and a ketonic oxygen as sole oxygenic substituent in ring C at the carbon atom 11.

3. A saturated etiocholanic acid, the rings A and B of which are linked together in cis-position, and which contains a hydroxyl at the carbon atom 3 and a ketonic oxygen as sole oxygenic substituent in ring C at the carbon atom 11.

4. B-hydroxy-ll-keto-etiocholanic acid methyl ester of melting point 170-174 C.

5. 3 hydroxy l1 keto etiocholanic acid of melting point 204207 C.

TADEUS REICHSTEIN.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,239,250 Robinson Apr. 22, 1941 2,255,264 Marker Sept. 9, 1941 2,255,265 Marker Sept. 9, 1941 2,265,488 Miescher Dec. 9, 1941 FOREIGN PATENTS Number Country Date 514,437 Great Britain Nov. 8, 1939 543,265 Great Britain Feb. 17, 1942 OTHER REFERENCES Steiger et al.: Helv. Chim. Acta, 21, 831, 840-841 (1938).

Sobotka: Chemistry of the Sterids, 1938,

page 164. 

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF A SATURATED ETIOCHOLANIC ACID AND THE CORRESPONDING SATURATED ETIOCHOLANIC ACID METHYL ESTER, THE RINGS A AND B OF WHICH COMPOUND ARE LINKED TOGETHER IN CIS-POSITION, AND WHICH COMPOUND CONTAINS A HYDROXYL AT THE CARBON ATOM 3 AND A KETONIC OXYGEN AS SOLE OXYGENIC SUBSTITUENT IN RING C AT THE CARBON ATOM
 11. 